Purine-2(3H),6(1H)-dione

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 99.9%

Analytical monitoring:

yes

Details on sampling:

- Sampling: collected at approximately 0 and 72 hours to measure concentrations of the test substance; at test initiation, samples collected from each test concentration and control solution prior to distribution into test chambers; at exposure termination, remaining biological replicates from each respective test concentration and control solutions pooled then sampled; at exposure termination, the abiotic control was sampled; samples collected in duplicate.
- Concentration (method): Concentrations in samples determined using a 4000 LC/MS/MS coupled with Infinity HPLC system.

Vehicle:

yes

Remarks:

freshwater AAP medium.

Details on test solutions:

0.63, 1.3, and 2.5 mg/L (dilutions of 5.0 mg/L stock with freshwater AAP medium)

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: freshwater green alga
- Strain: Raphidocelis subcapitata
- Source (laboratory, culture collection): Original algal cultures were obtained from the University of Waterloo on August 18, 2021.
- Method of cultivation: Algal cells used in this test were obtained from the testing facility cultures that had been actively growing in culture medium for at least two weeks prior to test initiation. Algal cells for this study were taken from a culture that had been transferred to fresh medium four days prior to test initiation

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

23.6-23.8°C

pH:

7.31-7.89 (test initiation)
7.82-8.01 (test termination)
7.52 (abiotic control)

Nominal and measured concentrations:

Nominal: 0.63, 1.3, 2.5, 5.0, 10 mg/L
Measured: 0.091, 0.13, 0.18, 0.26, 0.37 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile, 250-mL Erlenmeyer flasks plugged with sterile foam stoppers
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 100 mL
- Initial cells density: 3.46 X 10E6 cells/mL, determined using a hemacytometer and microscope. The target initial cell density of approximately 10000 cells/mL at test initiation was achieved by adding 0.289 mL of the culture to each replicate with a Sartorius digital pipette.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 1

GROWTH MEDIUM: The algal cells were cultured and tested in freshwater AAP medium. Stock nutrient solutions were prepared by adding reagent-grade chemicals to purified test facility well-water. The test medium was then prepared by adding appropriate volumes of the stock nutrient solutions to purified well water (NANOpure® water). The pH of the medium was adjusted to 7.5 ± 0.1 with 10% hydrochloric acid. The medium was sterilized by filtration (0.22 µm) prior to use.

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination
- Light intensity and quality: Cool-white fluorescent lighting at target intensity of 6000 ± 600 lux; light intensity measured using an LI-250A light meter with LI-210R photometric sensor at five locations surrounding test flasks on shaker tables at test initiation.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Test samples were collected from each of the biological replicates of the test concentrations and control for the determination of algal cell densities. Samples were collected at approximately 24-hour intervals during the 72-hour exposure, and samples were held in the dark under refrigerated conditions for a maximum of three days until cell counts were completed. Prior to counting, the sample solutions were sonicated for approximately 30 seconds and inverted prior and after dilution, if applicable, to ensure the cells within each sample were homogeneously distributed. Cell counts were performed using an electronic particle counter (Beckman Coulter® Z2 Series). Prior to conducting cell counts, the linearity of the instrument response was determined at settings previously established for Raphidocelis subcapitata. A primary counting standard containing Raphidocelis subcapitata cells was prepared at a nominal concentration of 100000 cells/mL, and the density was verified using a hemacytometer and a microscope. The standard was subsequently diluted to provide a series of seven counting standards for the determination of instrument linearity. Theoretical densities were assigned to each secondary counting standard based upon the verified density of the primary counting standard and the dilution ratio. The cell densities of the counting standards were measured using the electronic particle counter and were compared to the theoretical densities by performing a least squares regression analysis. Cell counts for samples collected during the test were conducted once instrument linearity was demonstrated (i.e., the R-squared values obtained through the regression analyses were at least 0.99933). A single aliquot of each sample collected during the test was diluted with an electrolyte solution (Isoton® II). Three 0.5-mL volumes of the diluted sample were counted, and the resulting counts were averaged. The cell density of the sample was determined by adjusting the mean cell count (cells/mL) obtained using the particle counter, based upon the Y-intercept and slope calculated through the regression analysis, and the dilution factor.
- Other: Microscopic examination of the inoculum culture was conducted prior to initiation of the test to assess whether the algae were normal in appearance. Samples of test solution were collected from each of the biological replicates per treatment and control group at the end of the exposure period. These samples were pooled within their respective treatments, and sub-samples were removed and examined microscopically for atypical cell morphology (e.g., changes in cell shape, size or color). Cells in the replicate test chambers also were assessed for aggregation, flocculation, or adherence of the cells to the test chamber.
PREPARATION OF TEST CONCENTRATIONS: A stock solution was prepared in freshwater AAP medium at a nominal concentration of 10 mg a.i./L. The 10 mg a.i./L stock was inverted at least twenty times and sonicated for approximately 1 hour. Another stock solution was prepared in freshwater AAP medium at a nominal concentration of 5.0 mg a.i./L. The solution was inverted at least twenty times and sonicated for approximately 1 hour to mix. The 10 mg a.i./L stock stirred until allotment into test vessels. The 5.0 mg a.i./L stock stirred while subsequent dilutions were prepared. Test solutions were prepared at target nominal concentrations of 0.63, 1.3, and 2.5 mg a.i./L by diluting aliquots of the 5.0 mg a.i./L stock with freshwater AAP medium. All test solutions appeared clear and colorless with no surface-slicks. Particulates were observed in all treatment groups. The negative control consisted of freshwater AAP medium without the addition of test substance. The abiotic control was an aliquot of the nominal 2.5 mg a.i./L test solution.

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 0.37 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

highest measured concentration tested which was at limit of water solubility

Basis for effect:

other: cell density, growth rate, and yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.37 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Remarks:

highest measured concentration tested which was at limit of water solubility

Basis for effect:

other: cell density, growth rate, and yield

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.37 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Remarks:

highest measured concentration tested which was at limit of water solubility

Basis for effect:

other: cell density, growth rate, and yield

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 0.37 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Remarks:

highest measured concentration tested which was at limit of water solubility

Basis for effect:

other: cell density, growth rate, and yield

Details on results:

The measured concentrations of test substance in matrix fortification samples ranged from 94.8 to 106% of nominal concentrations

Reported statistics and error estimates:

The calculation of cell densities, growth rates, yields and percent inhibition values, as well as all statistical analyses, were conducted using SAS Version 9.4. Yield was calculated for each treatment and control replicate at 72 hours as the final cell density after 72 hours of exposure minus the nominal initial cell density. Specific growth rates were calculated for each replicate of the control and test concentrations at each 24-hour interval of exposure using the following formula:

Specific Growth Rate: µ = (ln Nn - ln N0 / tn - t0) where:
µ = Average specific growth rate
N0 = Nominal cell density (cells/mL) at to
Nn = Measured cell density (cells/mL) at tn
tn = Time after beginning of test (hours)
to = Time of beginning of test (hours)

Cell density, growth rate and yield data were evaluated for normality and homogeneity of variance (α = 0.01) using the Shapiro-Wilk’s and Levene’s tests, respectively. The 72-hour cell density, growth rate data, and yield data met assumptions of normality and homogeneity of variance. The treatment responses were compared to the negative control response using Dunnett’s test (α = 0.05). The results of the statistical analyses, as well as an evaluation of the concentration-response pattern, were used to determine the NOEC values relative to each parameter at 72 hours. Inhibition values were calculated for each test concentration as the percent reduction in cell density, growth rate or yield relative to the negative control replicates using the following formula:

Percent Inhibition = ([Mean Control Response - Mean Treatment Response]/Mean Control Response) × 100

MEAN CELL DENSITY, GROWTH RATE, YIELD AND PERCENT INHIBITION DURING THE 72-HOUR EXPOSURE PERIOD
Geometric Mean, Measured Concentration (mg a.i./L)	0-72 Hour Cell Density^1		0-72 Hour Growth Rate^1		0-72 Hour Yield^1,3
	Mean Cell Density (cells/mL)*	Percent Inhibition^2	Mean Growth Rate (hour^-1)*	Percent Inhibition^2	Mean Yield (cells/mL)*	Percent Inhibition^2
Negative Control	1,068,684	--	0.0649	--	1,058,684	--
0.091	1,104,577	-3	0.0653	-1	1,094,577	-3
0.13	1,179,420	-10	0.0662	-2	1,169,420	-10
0.18	1,163,015	-9	0.0660	-2	1,153,015	-9
0.26	1,321,885	-24	0.0677	-4	1,311,885	-24
0.37	1,069,223	0	0.0648	0	1,059,223	0
^1Calculations were performed using SAS Version 9.4. Manual calculations may differ slightly. ^2Percent inhibition was calculated relative to the negative control replicates. ^3Yield was calculated as the final cell density (density at 72 hours) minus the initial cell density (10,000 cells/mL). * None of the treatment group means were significantly different (Dunnett’s test, p > 0.05) from the negative control mean.

MEASURED CONCENTRATIONS OF TEST SUBSTANCE IN TEST SOLUTION SAMPLES
Nominal Test Concentration (mg a.s./L)	Sample Number	Sampling Time (Day)	Measured Concentration^1,2 (mg a.s./L)	Percent of Nominal^2	Geometric Mean ± SD Measured Concentration ^3,5 (mg a.s./L)	Geometric Mean Measured Percent of Nominal^3
Negative Control (0.0)	1	0	<LOD	--	<LOD	--
	7	3	<LOD	--
0.63	2	0	0.666	106	0.091	14.5
	8	3	<LOD	--
1.3	3	0	1.29	99.2	0.13	9.77
	9	3	<LOD	--
2.5	4	0	2.52	101	0.18	7.10
	10	3	<LOQ	--
2.5 (abiotic)	4	0	2.52	101	2.6	103
	11	3	2.61	104
5.0	5	0	5.23	105	0.26	5.11
	12	3	<LOD	--
10	6	0	10.8	108	0.37	3.67
	13	3	<LOD	--
^1The limit of quantitation (LOQ) of the test substance in freshwater AAP medium was 0.0250 mg a.i./L, defined as the lowest nominal concentration of matrix fortification samples with a mean recovery within 70-120% and a relative standard deviation (RSD) ≤ 20%. The limit of detection (LOD) of the test substance in freshwater is set at 0.00400 mg a.i./L, defined as the lowest detectable concentration of an analyte in a sample and expressed as the lowest calibration standard concentration (0.00100 mg a.i./L) multiplied by the dilution factor of an LOQ sample (4.00). ^2Results were generated using Analyst Version 1.7.1. Manual calculations may differ slightly. ^3Results were generated using Excel 2016. Manual calculations may differ slightly. ^4Reanalysis of Day 3 samples confirmed original results. The original results are reported here. ^5 ½ the LOQ (0.0125 mg a.i./L) was used in calculations with a result of <LOD.

 

MEASURED CONCENTRATION OF TEST SUBSTANCE IN CENTRIFUGED TEST SAMPLE SOLUTION
Nominal Test Concentration (mg/ a.i./L)	Sample Number	Sampling Time (Day)	Measured Concentration^1,2 (mg a.i./L)	Percent of Nominal (%)^2
Negative Control (0.0)	1B	0	<LOD	--
0.63	2B	0	0.664	105
1.3	3B	0	1.35	104
2.5	4B	0	2.52	101
5.0	5B	0	5.16	103
10.0	6B	0	10.5	105
^1The limit of quantitation (LOQ) of the test substance in freshwater AAP medium was 0.0250 mg a.i./L, defined as the lowest nominal concentration of matrix fortification samples with a mean recovery within 70-120% and a relative standard deviation (RSD) ≤ 20%. The limit of detection (LOD) of the test substance in freshwater is set at 0.00400 mg a.i./L, defined as the lowest detectable concentration of an analyte in a sample and expressed as the lowest calibration standard concentration (0.00100 mg a.i./L) multiplied by the dilution factor of an LOQ sample (4.00). ^2Results were generated using Analyst Version 1.7.1. Manual calculations may differ slightly.

 

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour NOEC value for the test substance was determined to be > 0.37 mg a.i./L, highest measured concentration tested which was at limit of water solubility.

Executive summary:

The acute toxicity of the test substance to Raphidocelis subcapitata under static conditions was determined in a 72-hour exposure test. The test was conducted based on procedures outlined in the OECD guideline for the Testing of Chemicals, Guideline 201. The study was conducted with five concentrations of test substance, an abiotic control (at a mid-level test concentration), and a negative (freshwater AAP medium) control in an environmental chamber for three days at 24 ± 2⁰C. Three replicates were initiated for each test substance concentration. One replicate was prepared for the abiotic control. Six replicates were included in the negative control. Cell counts were taken from samples collected daily during the test from all biological replicates in the five treatment groups, and the six negative control replicates. Cell density and the corresponding yields and growth rates were determined. Nominal test concentrations selected were 0.63, 1.3, 2.5, 5.0, and 10 milligrams active ingredient per liter (mg a.i./L). Measured concentrations were determined from samples collected from each test concentration and negative control at the beginning of the test and from the pooled test solution of the remaining replicates at exposure termination using high performance liquid chromatography with tandem mass spectrometric detection (LC/MS/MS). Samples were also collected from one abiotic control (nominal 2.5 mg a.i./L) replicate at exposure termination. Samples collected for analytical verification at test initiation were analyzed in duplicate, with one set of samples centrifuged prior to analysis to remove particulates. Measured concentrations on day 0 in the non-centrifuged samples ranged from 99.2 to 108% of nominal. Measured concentrations on day 0 in the centrifuged samples ranged from 101 to 105% of nominal. Measured concentrations in the samples collected from the biotic replicates on day 3, pooled by experimental group were all less than the limit of detection. The measured concentration in the abiotic control at test termination was 104% of nominal. The nominal recovery at test termination in the abiotic replicate demonstrates that the test organism was driving the decrease in measured concentration over time in the biotic test solutions. At the end of the 72-hour exposure period, cell density increased in the negative control by at least a factor of 16 in three days. The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2, and 2-3) in the negative control replicates was 12.7%, which achieved the less than 35% criterion. The coefficient of variation of mean growth rate in the negative control replicates at test termination was 1.42%, which met the requirement of less than 7%. The 72-hour EC50, ErC50, and EyC50 values based on cell density, growth rate, and yield were empirically estimated to be > 0.37 mg a.i./L, highest measured concentration tested which was at limit of water solubility. The 24 and 48-hour EC50 and ErC50 values were empirically estimated to be > 0.37 mg a.i./L. The 72-hour NOEC value for cell density, growth rate, and yield was determined to be > 0.37 mg a.i./L, the highest concentration tested.  

Description of key information

OECD Guideline 201; green algae 72-hr ErC50 >0.37 mg/L (highest concentration tested, which was at limit of water solubility). Reliability = 1